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. 2014 Aug;111(8):1604-16.
doi: 10.1002/bit.25233. Epub 2014 May 22.

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool

Carlotta Ronda  1 , Lasse Ebdrup Pedersen, Henning Gram Hansen, Thomas Beuchert Kallehauge, Michael J Betenbaugh, Alex Toftgaard Nielsen, Helene Faustrup Kildegaard
Affiliations
Free PMC article

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool

Carlotta Ronda et al. Biotechnol Bioeng. 2014 Aug.
Free PMC article

Abstract

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.

Keywords: CRISPR Cas9; CRISPy; Chinese hamster ovary cells; database; genome editing.

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Figures

Figure 1
Figure 1
Genome editing in CHO cells by CRISPR Cas9. A: Schematics of the Cas9 and sgRNA expression cassettes. The Cas9 expression cassette consists of a CMV promoter, Cas9 ORF codon-optimized for CHO, SV40 nuclear localization sequence (NLS) and bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence. The sgRNA expression cassette consists of a U6 polymerase III promoter, a target gRNA sequence, a gRNA scaffold sequence and a poly(T) termination sequence. B: Illustration of the sgRNA genomic target sites in COSMC and FUT8. Red lines denote the position of the sgRNA target sites. Introns are depicted as broken lines (not drawn to scale) and exons as arrowed boxes. C: Indel frequency in COSMC analyzed by T7 endonuclease assay. Genomic DNA was extracted from CHO-K1 cells 5 days after transfection with plasmids encoding Cas9 and sgRNA against COSMC. The PCR amplicon covering the sgRNA-target sites as shown in panel B was re-annealed to enable heteroduplex formation before treatment with T7 endonuclease where indicated. Samples were subsequently analyzed by agarose gel electrophoresis. Approximate quantification of indels (%) was peformed with ImageJ software analysis of the uncut (WT) DNA bands. For details see Supplementary Table SVII.
Figure 2
Figure 2
Analysis of generated indels in COSMC. A: TOPO™ TA-based sequence analysis of COSMC. Genomic DNA was extracted from CHO-K1 cells 5 days after transfection with plasmids encoding Cas9 and sgRNA against COSMC. PCR amplicons covering the sgRNA-target sites in COSMC were TOPO™ TA-cloned and Sanger sequenced. Between 21 and 32 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table. B: Alignment of TOPO™ sequence traces. Genomic DNA from cells transfected with Cas9 + sgRNA1_C (replicate #1) were subjected to TOPO™ cloning as described in panel A. The sequence traces are aligned to the CHO-K1 genomic sequence. The red arrow indicates the genomic target site of sgRNA1_C. The numbers denote the position (bp) in the open reading frame of COSMC. Green, red and orange colors indicate insertions, deletions and substitutions, respectively. C: Targeted deep sequencing analysis of COSMC. The same extracted genomic DNA as described for panel A was used as template for the MiSeq analysis. Between 200,000 and 700,000 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table.
Figure 3
Figure 3
Functional and genomic knockout of FUT8 in CHO-K1. A: Targeted deep sequencing analysis of the FUT8 locus in CHO-K1 cells. Genomic DNA was extracted from CHO-K1 cells transfected with Cas9 and FUT8 sgRNAs harvested on Day 5 (no LCA selection) or Day 12 (7 days with LCA selection). The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table. B: Selection of FUT8 knockout CHO-K1 cells by LCA. As indicated, cells were either transfected with only a Cas9-encoding plasmid or in combination with an sgRNA3_F-encoding plasmid. Five days after transfection (Day 5), selection with LCA was initiated. The day after (Day 6), the shown bright field images were acquired. The magnified view shows cells with normal (adherent-looking) morphology from pool of cells transfected with Cas9 and sgRNA3_F. C: Phenotypic staining of FUT8 knockout CHO-K1 cells by fluorescein-labeled LCA (F-LCA). CHO-K1 cells were treated as described for panel A. On Day 13, cells were treated with Hoechst and with F-LCA where indicated. Fluorescence microscopy images were subsequently acquired. Hoechst and F-LCA signal is depicted as red and green color, respectively, in the merged images and as grayscale in the individual images. D: Quantification of fluorescent-based phenotypic staining of FUT8 knockout CHO-K1 cells. Cells were gated based on signal intensity of Hoechst and F-LCA as shown. The percentages of F-LCA positive (FUT8 WT) and negative (phenotypic knockout of FUT8) cells are shown. LCA: Lens culinaris agglutinin.
Figure 4
Figure 4
Cas9 activity in CHO results in high frequency of 1 bp indels. A: Frequency distribution of indel sizes. The size distribution of indels is based on sequences obtained from all eight sgRNAs. The indel frequency for each sgRNA is the average for both independent experiments. Normalization was performed so each of the eight sgRNA accounts for 12.5% of the data points. Only indels ranging from −37 bp (37 bp deletion) to +11 bp (11 bp insertion) are shown. B: Frameshifts generated by CRISPR Cas9. The distribution of indels generating ±1, ±2, and ±3 base pair shifts in the reading frame was calculated from data presented in panel A.
Figure 5
Figure 5
Workflow of Cas9 target finding tool: CRISPy. Screenshots of the online CRISPy tool showing the process of (1) finding a target gene, (2) selecting an exon, (3) evaluating the available targets, and (4) showing links to PCR primer designs for analysis of on- and off-target effects resulting from Cas9 + sgRNA activity. The tool is available at http://staff.biosustain.dtu.dk/laeb/crispy/.
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